Cardiomyocytes Sense Matrix Rigidity through a Combination of Muscle and Non-muscle Myosin Contractions

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Fibronectin rigidity response through Fyn and p130Cas recruitment to the leading edge

Cell motility on extracellular matrices critically depends on matrix rigidity, which affects cell adhesion and formation of focal contacts. Receptor-like protein tyrosine phosphatase alpha (RPTPalpha) and the alphavbeta3 integrin form a rigidity-responsive complex at the leading edge. Here we show that the rigidity response through increased spreading and growth correlates with leading edge recruitment of Fyn, but not endogenous c-Src. Recruitment of Fyn requires the palmitoylation site near the N-terminus and addition of that site to c-Src enables it to support a rigidity response. In all cases, the rigidity response correlates with the recruitment of the Src family kinase to early adhesions. The stretch-activated substrate of Fyn and c-Src, p130Cas, is also required for a rigidity response and it is phosphorylated at the leading edge in a Fyn-dependent process. A possible mechanism for the fibronectin rigidity response involves force-dependent Fyn phosphorylation of p130Cas with rigidity-dependent displacement. With the greater displacement of Fyn from p130Cas on softer surfaces, there will be less phosphorylation. These studies emphasize the importance of force and nanometer-level movements in cell growth and function.     

Opposing effects of PKCtheta and WASp on symmetry breaking and relocation of the immunological synapse

The immunological synapse (IS) is a junction between the T cell and antigen-presenting cell and is composed of supramolecular activation clusters (SMACs). No studies have been published on naive T cell IS dynamics. Here, we find that IS formation during antigen recognition comprises cycles of stable IS formation and autonomous naive T cell migration. The migration phase is driven by PKCtheta, which is localized to the F-actin-dependent peripheral (p)SMAC. PKCtheta(-/-) T cells formed hyperstable IS in vitro and in vivo and, like WT cells, displayed fast oscillations in the distal SMAC, but they showed reduced slow oscillations in pSMAC integrity. IS reformation is driven by the Wiscott Aldrich Syndrome protein (WASp). WASp(-/-) T cells displayed normal IS formation but were unable to reform IS after migration unless PKCtheta was inhibited. Thus, opposing effects of PKCtheta and WASp control IS stability through pSMAC symmetry breaking and reformation.     

Motor and cargo interactions.

The movements of intracellular cargo along microtubules within cells are often saltatory or of short duration. Further, calculations of the fraction of membrane vesicles that are moving at any period, indicate that active motor complexes are rare. From observations of normal vesicle traffic in cells, there appears to be position-dependent activation of motors and a balance of traffic in the inward and outward directions. In-vitro binding of motors to cargo is observed under many conditions but motility is not. Multi-component complexes appear to be involved in producing active organelle movements by a graded activation system that is highly localized in the cell. The basis of the activation of motility of the organelle motor complexes is still unknown but phosphorylation has been implicated in many systems. In the case of the motor-binding protein, kinectin, it has been linked to active organelle movements powered by conventional kinesin. From the coiled-coil structure of kinectin and the coiled-coil tail of kinesin, it is postulated that a coiled-coil assembly is responsible for the binding interaction. Many other cargoes are transported but the control of transport will be customized for each function, such as axonemal rafts or cytoskeletal complexes. Each function will have to be analyzed separately and motor activity will need to be integrated into the specific aspects of the function.     

Receptor-linked proteolysis of membrane-bound glucagon yields a membrane-associated hormone fragment

We have studied, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, glucagon-receptor complexes that arise from the incubation of canine hepatic plasma membranes with [[125I]iodo-Tyr10]glucagon. While a 54,000-dalton membrane protein was tentatively identified as the glucagon receptor by chemical cross-linking, an additional component having an apparent molecular weight of 30,000 was routinely identified as also resulting from glucagon-receptor interactions. The latter material, however, was not observed when gels were fixed prior to autoradiography and was not affected by the addition of cross-linking agents to membrane incubations. Subsequent analysis actually identified the material as a fragment of radiolabeled glucagon that contains at least residues 1-13, has no ability of its own to associate with plasma membranes, and remains tightly membrane bound once it has been formed by receptor-mediated processes. Formation of the fragment was inhibited by phenylmethylsulfonyl fluoride, glucagon, and GTP, but not by N-ethylmaleimide or by a variety of glucagon-related peptides. Overall, our results identify a proteolytic modification of glucagon this is linked to the binding of ligand to high affinity GTP-dependent receptors and the existence of a physically distinct state of receptor in which the binding site is tightly filled by a ligand fragment.     

Extension of filopodia by motor-dependent actin assembly.

A variety of mechanisms have been proposed to explain the forward extension of cytoplasm in advancing cells and axonal growth cones, including actin polymerization and osmotic swelling. Based on our observations of the filopodia of cultured neuronal growth cones, we propose a mechanism involving motor-induced extension and retraction. We observed that filopodia (actin-based protrusions 0.2-0.5 mu in diameter) extend and retract from growth cone lamellae at the same rate. Further, force is generated at the tips of filopodia which is sufficient to produce compressive buckling of the proximal portion of the filopodium. From our analysis of these movements we suggest that a motor protein powers both the extension and retraction of filopodia.     

Two activators of microtubule-based vesicle transport

Cytoplasmic dynein purified by nucleotide dependent microtubule affinity has significant minus end-directed vesicle motor activity that decreases with each further purification step. Highly purified dynein causes membrane vesicles to bind but not move on microtubules. We exploited these observations to develop an assay for factors that, in combination with dynein, would permit minus end-directed vesicle motility. At each step of the purification, non-dynein fractions were recombined with dynein and assayed for vesicle motility. Two activating fractions were identified by this method. One, called Activator I, copurified with 20S dynein by velocity sedimentation but could be separated from it by ion exchange chromatography. Activator I increased only the frequency of dynein-driven vesicle movements. Activator II, sedimenting at 9S, increased both the frequency and velocity of vesicle transport and also supported plus end movements. Our results suggest that dynein-based motility is controlled at multiple levels and provide a preliminary characterization of two regulatory factors.     

Single cytoplasmic dynein molecule movements: characterization and comparison with kinesin.

Cytoplasmic dynein is a major microtubule motor for minus-end directed movements including retrograde axonal transport. To better understand the mechanism by which cytoplasmic dynein converts ATP energy into motility, we have analyzed the nanometer-level displacements of latex beads coated with low numbers of cytoplasmic dynein molecules. Cytoplasmic dynein-coated beads exhibited greater lateral movements among microtubule protofilaments (ave. 5.1 times/microns of displacement) compared with kinesin (ave. 0.9 times/micron). In addition, dynein moved rearward up to 100 nm over several hundred milliseconds, often in correlation with off-axis movements from one protofilament to another. We suggest that single molecules of cytoplasmic dynein move the beads because 1) there is a linear dependence of bead motility on dynein/bead ratio, 2) the binding of beads to microtubules studied by laser tweezers is best fit by a first-order Poisson, and 3) the run length histogram of dynein beads follows a first-order decay. At the cellular level, the greater disorder of cytoplasmic dynein movements may facilitate transport by decreasing the duration of collisions between kinesin and cytoplasmic dynein-powered vesicles.     

Sublethal damage during cryopreservation of rainbow trout sperm

Although cellular damage during cryopreservation of freshwater fish spermatozoa has been reported in several studies, there is a lack of correlation between this damage and the fertility rates of eggs using postthawed milt. The apparent lack of such correlation may be due to other undetected sublethal cryodamage, which could affect the cell functionality and viability. This may be extremely important for freshwater fish spermatozoa whose ability to fertilize the egg requires dilution in water or hypoosmotic solutions, an hazardous environment for the cells. This study tested the change in cell permeability during cryopreservation, using Hoechst 33258 to assess cell permeability. The permeability of spermatozoa at different times after dilution in several hypoosmotic media were investigated. In the first trial, fresh semen, sperm diluted in freezing media (CPT), and freeze/thawed semen were studied. Three CPT were tested (Me2SO, DMA, and methanol). In the second trial, the addition of egg yolk as a membrane stabilizer was investigated. Samples were frozen at -20 degreesC/min in a programmable cooler and thawed in a 25 degreesC water bath. Dilution in the CPTs slightly increased the susceptibility of cells to damage but freezing/thawing caused a dramatic increase in the fragility of cells, which were killed in a few seconds after their contact with the hypoosmotic solutions. Egg yolk provided a significant protection to the membrane, allowing the cells a greater and more prolonged survival in the fertilization media. Samples frozen with Me2SO displayed the best results. These results are consistent with the achieved fertility rates that demonstrated sublethal cryodamage in the function of the sperm membrane that was not detected by standard procedures. Copyright 1998 Academic Press.